System and method for irradiating a sample

ABSTRACT

Method and system for footprinting a nucleic acid molecule. A sample consists of a nucleic acid molecule in an environment in which —OH radicals are generated when the environment is irradiated with an X-ray beam having an intensity less than 10 9  photons sec −1  mm −2  for an amount of time less than 1,000 msec. The sample is then irradiated with an X-ray beam having an intensity less than 10 9  photons sec −1  mm −2  for an amount of time less than 1,000 msec so as to generate —OH radicals in the environment. Fragmentation of the nucleic acid molecule is then detected.

FIELD OF THE INVENTION

This invention relates to biophysical methods, and more specificallyrelates to such methods for irradiating a sample, particularly a samplecontaining a biological molecule.

PRIOR ART

The following is a list of prior art which is considered to be pertinentfor describing the state of the art in the field of the invention.Acknowledgement of these references herein will be made by indicatingthe number from their list below within brackets.

REFERENCES

-   1. Tullius, T. D. (1989) Physical studies of protein-DNA complexes    by footprinting. Annu Rev Biophys Biophys Chem, 18, 213-37.-   2. Brenowitz, M., Senear, D. F., Shea, M. A. and    Ackers, G. K. (1986) Quantitative DNase footprint titration: a    method for studying protein-DNA interactions. Methods Enzymol, 130,    132-81.-   3. Galas, D. J. and Schmitz, A. (1978) DNAse footprinting: a simple    method for the detection of protein-DNA binding specificity. Nucleic    Acids Res, 5(9), 3157-70.-   4. Tullius, T. D. and Dombroski, B. A. (1986) Hydroxyl radical    “footprinting”: high-resolution information about DNA-protein    contacts and application to lambda repressor and Cro protein. Proc    Natl Acad Sci USA, 83(15), 5469-73.-   5. Tullius, T. D., Dombroski, B. A., Churchill, M. E. and    Kam, L. (1987) Hydroxyl radical footprinting: a high-resolution    method for mapping protein-DNA contacts. Methods Enzymol, 155,    537-58.-   6. Strahs, D. and Brenowitz, M. (1994) DNA conformational changes    associated with the cooperative binding of cI-repressor of    bacteriophage lambda to OR. J Mol Biol, 244(5), 494-510.-   7. King, P. A., Jamison, E., Strahs, D., Anderson, V. E. and    Brenowitz, M. (1993) ‘Footprinting’ proteins on DNA with    peroxonitrous acid. Nucleic Acids Res, 21(10), 2473-8.-   8. Burkhoff, A. M. and Tullius, T. D. (1987) The unusual    conformation adopted by the adenine tracts in kinetoplast DNA. Cell,    48(6), 935-43.-   9. Latham, J. A. and Cech, T. R. (1989) Defining the inside and    outside of a catalytic RNA molecule. Science, 245(4915), 276-82.-   10. Celander, D. W. and Cech, T. R. (1990)    Iron(II)-ethylenediaminetetraacetic acid catalyzed cleavage of RNA    and DNA oligonucleotides: similar reactivity toward single- and    double-stranded forms. Biochemistry, 29(6), 1355-61.-   11. Franchet-Beuzit, J., Spotheim-Maurizot, M., Sabattier, R.,    Blazy-Baudras, B. and Charlier, M. (1993) Radiolytic footprinting.    Beta rays, gamma photons, and fast neutrons probe DNA-protein    interactions. Biochemistry, 32(8), 2104-10.-   12. Isabelle, V, Franchet-Beuzit, J., Sabattier, R.,    Spotheim-Maurizot, M. and Charlier, M. (1994) Sites of strand    breakage in DNA irradiated by fast neutrons. Biochimie, 76(2),    187-91.-   13. Sclavi, B., Woodson, S., Sullivan, M., Chance, M. R. and    Brenowitz, M. (1997) Time-resolved synchrotron X-ray “footprinting”,    a new approach to the study of nucleic acid structure and function:    application to protein-DNA interactions and RNA folding. J Mol Biol,    266(1), 144-59.-   14. Klassen, N. V. (1987), Radiation Chemistry Principles &    Applications. VCH, Taxes, pp. 29-61.-   15. von Sonntag, C. (1991) The chemistry of free-radical-mediated    DNA damage. Basic Life Sci, 58, 287-317; discussion 317-21.-   16. Hayes, J. J., Kam, L. and Tullius, T. D. (1990) Footprinting    protein-DNA complexes with gamma-rays. Methods Enzymol, 186, 545-9.-   17. Sclavi, B., Woodson, S., Sullivan, M., Chance, M. and    Brenowitz, M. (1998) Following the folding of RNA with time-resolved    synchrotron X-ray footprinting. Methods Enzymol, 295, 379-402.-   18. Hampel, K. J. and Burke, J. M. (2001) Time-Resolved    Hydroxyl-Radical Footprinting of RNA Using Fe(II)-EDTA. Methods,    23(3), 233-239.-   19. Ralston, C. Y, He, Q., Brenowitz, M. and Chance, M. R. (2000)    Stability and cooperativity of individual tertiary contacts in RNA    revealed through chemical denaturation. Nat Struct Biol, 7(5),    371-4.-   20. Petri, V. and Brenowitz, M. (1997) Quantitative nucleic acids    footprinting: thermodynamic and kinetic approaches. Curr Opin    Biotechnol, 8(1), 36-44.-   21. Tullius, T. D. and Dombroski, B. A. (1985) Iron(II) EDTA used to    measure the helical twist along any DNA molecule. Science,    230(4726), 679-81.-   22. Yang, S. W. and Nash, H. A. (1994) Specific photocrosslinking of    DNA-protein complexes: identification of contacts between    integration host factor and its target DNA. Proc Natl Acad Sci USA,    91(25), 12183-7.-   23. Nelson, W. R., Hirayama, H. and Rogers, D. W. O. (1985), pp.    265.-   24. Namito, Y., Ban, S. and Hirayama, H. (1995) Phys. Rev., 51,    3036-3043.-   25. Namito, Y., Ban, S. and Hirayama, H. (1993) Implementation of    linearly-polarized photon scattering into the EGS4 code. Nucl.    Instrum. Methods Phys. Res, 332, 277-283.-   26. Dixon, W. J., Hayes, J. J., Levin, J. R., Weidner, M. F.,    Dombroski, B. A. and Tullius, T. D. (1991) Hydroxyl radical    footprinting. Methods Enzymol, 208, 380-413.-   27. Tullius, T. D. (1991) DNA footprinting with the hydroxyl    radical. Free Radic Res Commun, 12-13 Pt 2, 521-9.-   28. Pastor, N., Weinstein, H., Jamison, E. and Brenowitz, M. (2000)    A detailed interpretation of OH radical footprints in a TBP-DNA    complex reveals the role of dynamics in the mechanism of    sequence-specific binding. J Mol Biol, 304(1), 55-68.-   29. Ralston, C. Y, Sclavi, B., Sullivan, M., Deras, M. L.,    Woodson, S. A., Chance, M. R. and Brenowitz, M. (2000) Time-resolved    synchrotron X-ray footprinting and its application to RNA folding.    Methods Enzymol, 317, 353-68.-   30. Rice, P. A., Yang, S., Mizuuchi, K. and Nash, H. A. (1996)    Crystal structure of an IHF-DNA complex: a protein-induced DNA    U-turn. Cell, 87(7), 1295-306.-   31. Dhavan, G. M., Crothers, D. M., Chance, M. R. and    Brenowitz, M. (2001) Concerted Binding and Bending of DNA by    Eschericia coli Integration Host Factor. Submmited.-   32. Hubell, J. H., Veigele, W. J., Briggs, E. A., Brown, R. T.,    Cromer, D. T. and Howerton, R. J. (1975) Atomic Form Factors,    Incoherent Scattering Functions, and Photon Scattering Cross    Sections. J. Phys. Chem. Ref. Data, 4, 471.-   33. Wu, J. C., Kozarich, J. W. and Stubbe, J. (1983) The mechanism    of free base formation from DNA by bleomycin. A proposal based on    site specific tritium release from Poly(dA.dU). J Biol Chem, 258(8),    4694-7.-   34. Heyduk, T., Baichoo, N. and Heyduk, E. (2001) Hydroxyl radical    footprinting of proteins using metal ion complexes. Met Ions Biol    Syst, 38, 255-87.

BACKGROUND OF THE INVENTION

Interactions between proteins and nucleic acids and nucleic acidconformations are commonly examined by “footprinting” methods. In thesemethods, a nucleic acid molecule is fragmented by applying an agent thatproduces nicks in the phosphodiester backbone of the molecule. Regionsof the molecule devoid of nicks are then sought. Such regions devoid ofnicks are regions that were protected from the effects of the agent.Such protection may be due, for example, to binding of a ligand to aspecific sequence of bases in the nucleic acid or to the conformation ofthe molecule^((1, 2)). The prerequisite of such assays is the ability toproduce and detect high-quality nucleic acid fragmentation. Nucleic acidfragmentation can be achieved by using a variety of enzymatic andchemical reagents⁽³⁾. Another method of nucleic acid fragmentationreferred to as “chemical hydroxyl radical footprinting” uses Fentonchemistry⁽⁴⁻⁶⁾ and peroxonitrous acid⁽⁷⁾. In this method, hydroxylradicals (—OH) engender breaks of the phosphodiester backbone in anon-specific sequence manner. Using hydroxyl radical methods overenzymatic footprinting is advantageous because it provides greatsensitivity to nucleic-acid structures, such as sequence-dependentcurvature⁽⁸⁾ and RNA folding⁽⁹⁾.

Nucleic acid cleavage by hydroxyl radical is predominantly dependentupon the solvent accessibility of the phosphodiester backbone.Additionally, it is relatively insensitive to base sequence, and it isnot important whether the nucleic acid is single or double stranded⁽¹⁰⁾.—OH can be generated by Fe-EDTA catalysis or by γ-rays, β particles,fast neutrons, and X-ray radiation⁽¹¹⁻¹³⁾. The radiolysis of water byX-rays with energies from 100 eV up to the MeV range produces freeelectrons and —OH according to the overall reaction illustrated inequation (1)⁽¹⁴⁾.

The —OH generated by this reaction can abstract a hydrogen from the C′4carbon of the ribose sugar of DNA and RNA, leading to breakage of thephosphodiester backbone of the polymer⁽¹⁵⁾. Controlled exposure ofprotein-nucleic acid complexes to X-rays has been used to detectspecific interactions within such complexes^((11, 16)). X-ray mediatedfootprinting has been shown to be an attractive method for time-resolvedfootprinting studies, because it can produce a high flux of —OH thatfragments the nucleic acid backbone in millisecond time scales withbasepair resolution. The recent development of synchrotron time-resolvedX-ray footprinting demonstrated the utilization of this method to studydynamic structural changes in RNA folding⁽¹⁷⁾.

High radiation sources like synchrotron can produce a sufficiently highphoton flux to generate a sufficient concentration of —OH radicals tofragment nucleic acids within of tenths of milliseconds⁽¹³⁾. However,the use of a synchrotron radiation source in footprinting is limited dueto its relative inaccessibility and the high cost of its operation.

SUMMARY OF THE INVENTION

In its first aspect, the present invention provides a system forirradiating a sample. In accordance with this aspect of the invention,the system comprises an X-ray generator that generates ax X-ray beamhaving an intensity less than 10⁹ photons sec⁻¹ mm⁻². For example, arotating anode may be used in the system. The system also comprises ashutter capable of alternating between open and closed states such thatthe shutter may be in the open state for a predetermined amount of timeless than 1,000 msec. In its second aspect, the invention provides amethod for irradiating a sample. In accordance with this aspect of theinvention, a sample is irradiated with an X-ray beam having an intensityless than 10⁹ photons sec⁻¹ mm⁻². The sample is exposed to the radiationfor an amount of time less than 1000 msec.

The invention may be used, for example, to irradiate a sample containingnucleic acid molecules so as to generate —OH radicals in the sample andto fragment the molecules. This allows —OH radical footprinting of DNAto be performed in time scales suitable for studying the structuralkinetics of RNA folding⁽¹⁹⁾ and protein-nucleic acid interactions⁽²⁰⁾.

The invention provides a system for irradiating a sample comprising:

-   -   (a) an X-ray generator capable of generating an X-ray beam        having an intensity less than 10⁹ photons sec⁻¹ mm⁻²; and    -   (b) a shutter alternating between an open state in which the        sample is irradiated by the X-ray beam and a closed state in        which the sample is not irradiated by the X-ray beam, the        shutter being configured to be in the open state for an amount        of time less than 1,000 msec.

The invention still further provides a method for irradiating a sample,comprising irradiating the sample with an X-ray beam having an intensityless than 10⁻⁹ photons sec⁻¹ mm⁻²m and for an amount less than 1,000msec.

Yet still further the invention provides a method for footprinting anucleic acid molecule comprising:

-   -   (a) placing the nucleic acid molecule in an environment in which        —OH radicals are generated when the environment is irradiated        with an X-ray beam having an intensity less than 10⁹ photons        sec⁻¹ mm⁻² for an amount of time less than 1,000 msec;    -   (b) irradiating the environment with an X-ray beam having an        intensity less than 10⁹ photons sec⁻¹ mm⁻² for an amount of time        less than 1,000 msec so as to generate OH radicals in the        environment;    -   (c) detecting fragmentation of the nucleic acid molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, a preferred embodiment will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIG. 1 shows a system for irradiating a sample in accordance with oneembodiment of the invention;

FIG. 2 shows Monte Carlo simulations of the absorbed energy in a DNAsample by different rotating anode target elements;

FIG. 3 shows a dose-response curve of beam intensities vs. fraction ofuncleaved DNA;

FIG. 4 shows DNA fragmentation by Rotating X-ray Anode in basepairresolution: (a) A basepair resolution portrait of 140 bp DNA fragmentafter exposure to the X-ray beam, (b) Peak fitting demonstration of thebands intensities resulted from the cleavage pattern of the DNA. Thedata analysis was performed as described by Pastor et al⁽²⁸⁾. The uppergraph represents raw data obtained from the program ImageQuant 5.0 (MD)and the lower graph presents the data after performing peak fitting;

FIG. 5 shows a dose-response curves relating the amount of uncleaved DNAto the time of sample exposure to the X-ray source, controlled by amilliseconds shutter;

FIG. 6 shows footprinting of IHF-DNA complex: (a) the IHF binding DNAsequence, (b) 10% sequencing gel of 165 bp DNA ³²P-labeled, (c)containing a 34 bp region of IHF binding sequence;

FIG. 7 shows the normalized photon flux spectrum at the X-9 NSLS beamline compared with the normalized absorbed energy distribution on anucleic acid sample in aqueous solution; and

FIG. 8 shows parameters of the rotating anode.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

FIG. 1 shows a schematic representation of a system for irradiating asample, generally designated by 100, in accordance with one embodimentof the invention. The system comprises a model RTP 500 rotating anodetube 110 purchased from Rigaku, Japan. The rotating anode machine 110generates an X-ray beam 115 that irradiates a sample 120. As explainedbelow, in the embodiment of FIG. 1, the sample 120 is either placed atthe bottom of an Eppendorf tube 122 or on the cover of an Eppendorf tube124. The Eppendorf tube 122 or 124 is then placed in a sample holder 125which in turn is inserted into an adapter 128 so as to position thesample in the X-ray beam 115.

In accordance with the invention, the system 100 comprises an electronicshutter 130 purchased from Vincent Associates, Rochester, N.Y. Theelectronic shutter 130 is used for setting exposure times of up to 1,000msec. The electronic shutter 130 is preferably kept in a vacuum untilthe X-rays exit the anode tube 110. In order to further minimize thedistance that the X-ray beam travels in air between the anode tube 110and the sample, the sample is preferably placed in the cap of theEppendorf tube 124. The electronic shutter 130 is controlled through acable 140 from a remote control box 145 that was purchased from VincentAssociates, Rochester, N.Y. The electronic shutter was rigorously testedfor X-ray resistance.

The system 100 may optionally have a manual mechanical shutter 150 thatmay be used for setting exposure times of over 1 sec. When the manualshutter 150 is used, the sample 120 is placed at the bottom of anEppendorf tube 122. The Eppendorf tube is then placed in the adapter 125with the sample positioned in the X-ray beam 115. In the embodiment ofFIG. 1, the X-ray beam 115 travels 3.6 cm in air from the manual shutter150 to the bottom of the Eppendorf tube 122.

All of the inserted devices are preferably made of brass. This allowsdirect irradiation of the sample with the X-ray beam without anydivergence.

The system of FIG. 1 was used to perform footprinting of DNA moleculesas described in the following examples.

Materials and Methods

DNA cloning and plasmid purification—A random fragment from E. coliisolated chromosomal DNA, restricted with BamHI and EcoRI restrictionenzyme, was cloned into pre-digested pTZ18R (Amersham Pharmacia Biotech)with the same enzymes. After transforming E. coli XL-1 Blue strain(Stratagene), the plasmid was amplified and purified by using a concertnucleic acid purification system kit (GibcoBRL). A DNA fragment was cutby enzymatic restriction to produce a fragment of 140 bp; it waspurified with a Millipore-DNA purification kit from 1% agarose gel.

Preparation and end labeling of DNA molecules—DNA was end labeled withKlenow fragment (Roch Molecular Biochemicals) with α-[³²P]-ATP usingstandard protocol. The labeled DNA was loaded into 8% PAGE/Tris BorateEDTA (TBE) X 1.0 native gel and the DNA fragment was excised from thegel. The DNA was extracted from the gel by electroelution using theSchleicher & Schuell BIOTRAP system. DNA was precipitated andresuspended in Cacodylic buffer pH 7.5 10 mM.

Chemical footprinting—Fe (II)-EDTA cleavage reactions were performed bythe method of Tullius and Dombroski (21). Cleavage reaction components,1 μl of 0.2% H₂O₂, 1 μl of 60 mM sodium ascorbate, and 1 μl Fe (II)-EDTA(60 mM Fe (NH₄)₂—(SO₄)₂ and 50 mM Na₂-EDTA, pH 8 mixed prior to chemicalreaction) were added sequentially to the inside wall of an Eppendorftube. Rapid mixing was then applied by tapping the Eppendorf tube on asurface. Reactions were allowed to proceed for 2 sec and then quenchedwith 90 μl of 100% ethanol, 0.3 M NaACc pH 5.2, 0.4 μg tRNA,precipitated in −20° C. and resuspended in loading buffer.

X-ray footprinting—DNA footprinting was performed in siliconizedEppendorf tubes with a specific activity of 60,000 cpm. 10 μl samples oflabeled DNA (60,000 cpm specific activity) were incubated on ice priorto exposure to X-rays. Each sample was placed and exposed to X-raysusing the device of the invention. The X-ray shutter was open for theindicated time windows. Immediately after exposure to X-rays, thesamples were put on ice and combined with an equal volume of loadingbuffer: 90% formamide, 0.5×TBE buffer, 1 mM EDTA and 0.05 (w/v) xylenecyanole. Samples were kept at −80° C. for further analysis.

Protein-DNA footprinting—Purified Integration host factor M) proteinfrom E. coli and its DNA substrate were provided by Dr. MichaelBrenowitz and Gurie Dhavan. IHF was stored in Tris-HCl pH 7.5, 200 mMNaCl, 1 mM EDTA and 5% glycerol. The target DNA substrate of IHF (FIG. 5a) was prepared by PCR amplification of a plasmid carrying IHF consensussequence⁽²²⁾. The resulting PCR product of 164 bp was prephosphorylatedin one of its primers with cold ATP before the PCR reaction. The secondphosphorylation was performed after the PCR with ³²P-γ-ATP, usingstandard T4 polynucleotide kinase (NEB). DNA was electroeluted asdescribed above. Footprinting reactions were performed in 20 mMCacodylate buffer at pH 7.5, 50 mM KCl, 1 mM MgCl₂, at room temperaturefor 10 min. The reaction was quenched by ethanol precipitation,resuspended in loading buffer, and analyzed by gel electrophorsis.

Gel electrophoresis—Samples were applied to a pre-warm 10% denaturingpolyacrylamide-sequencing gel. Electrophoresis was performed at 50° C.buffered with 1.0×TBE. After completing the run, the gels were fixedwith 10% acetic acid and 10% methanol for 20 min, followed by incubationof the gel in 10% glycerol for 20 min. Autoradiographs of the dried gelswere analyzed by densitometry using Molecular Dynamics (MD)PhosphoImager Storm 820.

Data analysis of footprinting experiments—Data was analyzed withImageQuantNT software, (MD). Quantification of the fraction thatremained uncut was calculated according the following equation:

$F = \frac{{OD}_{n,{cut}}}{{OD}_{uncut}}$

where P equals the uncut fraction, OD_(n, cut) is the measured intensityof the n^(th) band, and OD_(uncut) is the unexposed sample measuredintensity. The data were fitted to a semi-logarithmic plot toexponential decay 1^(st) order: Y=y₀+Ae^(−x/t). Each data point is anaverage of two independently performed experiments.

Monte Carlo theoretical simulations of the photon absorption—The lastdevelopments in the EGS4 code system (23) for low energy scattering andpolarization established the code system as one of the best tools forsynchrotron-based X-ray simulations. The modular structure of the lowscattering photon transport routines, developed by the KEK EGS group(24,25), enables preparation of compound cross sections with formfactors and scattering functions as H₂O, air, DNA (as a polymer ofC₅O₅H₇—P), and polypropylene. The EGS4 system was used to simulate thedeposited energy in the footprinting samples for different photonsources. The first user-code was developed to include the NationalSynchrotron Light Source (NSLS) beam line X9-. A spectrum shape wascalculated by information found on the web(http://www-cxro.lbl.gov/optical_constants/bend2.html). Conditions usedfor calculation were 92% linear polarization ratio, introducing the DNAsample inside the conic-shaped end of a polypropylene “Eppendorf” tube(1.5 ml), sealed by a 1 mm cover made of the same material. The absorbedenergies per incident flounce in the cover and in the sample were scoredto find the efficiency of the NSLS photon source for footprinting.

Another group of user-codes was written for the X-ray generatorfootprinting irradiation system, which included the filtration (Be, andair), the source energy due to the anode material, and the sample insidethe opened polypropylene cylinder (as shown in FIG. 2). The same outputswere applied in the X-ray generator simulations as in the NSLSsimulation, with 10⁷ histories for each.

Results

Fragmentation of nucleic acid molecules has been shown to be dependentupon the effective concentration of free radicals, which are generatedfrom water radiolysis⁽²⁶⁾. In the case of X-ray footprinting, theconcentration of free radicals in solution is controlled by monitoringthe X-ray exposure (“dose”) received by the DNA/RNA sample. Theexperimentally determined dose-response curve provides the requiredX-ray exposure time in which each DNA/RNA molecule is cleaved only once.X-ray exposures resulting in 10-30% of the nucleic acid cleavageimplement this requirement. The sample preparation procedure and thebuffer system used in these experiments were optimized by conductingdose-response experiments for the different reaction conditions.

Sample preparation—Factors that contribute to —OH radical scavengingwere minimized in order to increase the effective concentration of thefree radicals in solution. The purification of labeled DNA is usuallyconducted by extraction from the polyacrylamide gel matrix. Thisprocedure introduces polyacrylamide contamination in the extractedDNA/RNA solution. Therefore, the DNA from the gel matrix was purified byelectroelution. This method remarkably lowered the required exposuretime of the nucleic acid sample to the X-ray beam to achieve 10-30% ofnucleic acid cleavage. In addition, three buffer systems were tested (pH7.5, 10 mM): Tris-HCl, HEPES-Na, and Cacodylic-Na. The best results wereobtained with the Cacodylic buffer. This buffer system is used often inX-ray footprinting procedures^((13,18)). Overall, these results showthat the sample composition and purification play a key role in theX-ray footprinting assay generated by the rotation anode machine.

Rotating anode parameters—The photon flux intensity and the energy ofthe rotating anode X-ray source were optimized to achieve maximum yieldof nucleic acid fragmentation. This was done using both theoretical andexperimental studies. The photon flux generated by the X-ray beam can becalculated for a given operation power and material target. The photonspectral flux for common diagnostic X-ray generators was calculated andsummarized in a catalogue (Birch et al, 1979) for a variety of anodematerials and tube voltages. The referenced flux value per Amp wasmultiplied with the rotating anode current to calculate the photon fluxexiting the source. The flux-per-amp value of 1.91×10^(6 ph mA)⁻¹s⁻¹mm⁻² for the molibdium (Mo) target, at 50 kV tube voltage, wasselected from the catalogue as a reference value without addingfiltration, because the filtration was included in the EGS4 Monte Carlouser-code simulation. The source photon flux for a Mo rotating-anode(300 mA) is, therefore, 5.7×10⁸ ph s⁻¹mm⁻² at 17.48 keV. The photon fluxof other low Z-number target materials (such as Copper (Cu), Chromium(Cr), Nickel, (Ni), Iron (Fe) anodes) at the K_(α) emission lines can becompared, due to the Mo florescence yields ratios. These results showthat the photon flux generated from rotating anode is one order ofmagnitude lower than the photon flux used in synchrotron footprintingexperiments in which a 200 mA ring current generates 5×10⁹ ph s⁻¹⁽¹⁷⁾.

To calibrate the specific beam energy with the shortest time scalesrequired for effective nucleic footprinting, three different targetelements were tested as anodes. The effect of the X-ray energy source onthe amount of DNA cut was examined at a given time by conductingdose-response experiments. In this experiment silver (Ag), Mo, and Cutarget anodes were used, which represent a relatively wide range ofenergy: (Ag, λ=0.56 Å (25.5 KeV); Mo, λ=0.71 Å (19.9 KeV); Cu, λ=1.54 Å(8.97 KeV)).

The results of these experiments, as well the calculated values of thephoton flux generated from each anode and the percent energy fractionabsorbed in the sample, are summarized in the table of FIG. 8. Eachresult is the average of two independent samples. The Monte Carlotheoretical simulation was conducted as described above in Materials andMethods.

FIG. 8 shows that the Cu anode achieved the most efficient DNAfragmentation, with the given X-ray generator setup, because most of thegenerated energy is deposited in the sample. Furthermore, the resultsshow that the main factor of efficient DNA cleavage is the amount ofenergy absorbed in the sample, which is correlated by the specificenergy of the source (FIG. 8). To further examine the effect of beamenergy on DNA fragmentation, Monte Carlo calculations were performed. Inthe case of X-ray generators with different anode metals, the absorbedenergy resulting from the Monte Carlo simulations was introduced tooptimize the rotating anode selection due to the maximum sampleradiation absorption. The fluorescence yield of each material wasmultiplied by the flux to compare the absolute sample absorptions (seealso Table 1). FIG. 2 shows the simulation results of sample absorptionefficiency versus the anode material Z-number. These results show thatthe energy at KeV between 7-9.5 contributes the most to thefragmentation of DNA in aqueous solution. This range of energy isconsistent with the energy of a Cu rotating anode (9 KeV). Therefore,the Cu rotating anode was found to be the most appropriate material foruse with X-ray generator-based footprinting.

X-ray beam intensity—The beam intensity parameter and its relation topercent DNA cut was examined. DNA samples of ˜50,000 cpm were exposed tothe X-ray beam with Cu target source for 3 seconds at different beamintensities Kw=KeV·mAMPS). DNA samples were analyzed by UREA/PAGEelectrophoresis. The top band on the gels, representing the full-lengthnucleic acid molecules, was quantified as described in Materials andMethods. The data sets represent two independent determinations.Fragmentation of DNA shows linear relationship to the intensity of theX-ray generator. The linear fit was extrapolated to higher intensity toshow the effect of the beam flux (intensity) on the fragmentation of DNA(R²=0.98). FIG. 3 shows the relationship between the intensity of theX-ray beam and the fraction of uncut DNA after exposure to X-ray. Theseresults show that the intensity of the X-ray beam exhibits a linearrelationship with the logarithm of the amount of fraction of DNAfragmented. Theoretical extrapolation of the linear line indicates thathigher beam intensities resulted in increased percent of DNAfragmentation in less time. Thus, a combination of the appropriatesource energy with the highest intensity that can be achieved by theX-ray generator contributes to maximum yield of DNA fragmentation inminimal time scales.

Resolving DNA at basepair resolution—Following nucleic acidfragmentation at basepair resolution requires the nucleic acidfragmentation method to show no discrepancy in cutting preferences. Inaddition, the electrophotogram of the nucleic acid fragments should beable to resolve long DNA/RNA molecules with high resolution of bandseparation. Hydroxyl radical had been shown to produce such a pattern ofcleavage from both chemical and X-ray sources^((13, 27)).

FIG. 4 a shows the portrait of 140 bp DNA fragment after exposure to therotating anode X-ray beam for 1 to 4 seconds. The digital image wasobtained by phosphor storage imaging of a 12% UREA/PAGE wedge-spaced gelof the reaction products resulting from a 1-4 seconds exposure to theX-ray beam of a 140 bp DNA ³²P-labeled restriction fragment labeled. Thelane designated FeEDTA was treated with the Fenton reaction on the sameDNA preparation, but was analyzed on a separate gel using the sameconditions as describe above. The insert is an enlargement of theindicated area. The results were comparable to the DNA cleavage patternsobtained by FeEDTA protocols⁽¹⁸⁾. The X-ray rotating anode footprintingin FIG. 4 shows exceptionally uniform and reproducible cleavage patterns(see insert). The integrated band intensity analysis (FIG. 4 b) shows ahighly homogeneous pattern throughout a long region of separated gelbands⁽²⁸⁾. To better resolve the DNA fragments during electrophoresis,we used wedge gel spacers, which increase the length of separation ofnucleic acid as well as the uniformity of the bands. With thisarrangement, up to 120 base pairs of a DNA molecule can be resolved withsufficient quality to perform quantitative footprinting analysis. Tominimize the path traveled by the X-ray beam in air, the Eppendorf covercap was used to hold the sample as close as possible to the end of thevacuum line facing the exit of X-rays. With this improved arrangement,the time required to achieve 10-30% DNA cleavage of a given DNA fragmentis lowered to the millisecond time regime.

FIG. 5 a shows the dose-response results of the 140 bp DNA fragment.Quantification of the fraction uncleaved was calculated as described inMaterials and Methods. The results are an average of two independentdeterminations. The percent DNA cut was measured after exposure to theX-ray source in milliseconds time scales. The dose-response densitometryanalysis shows that 10% cleavage occurs in 300 milliseconds (Cu, 60 KeV,300 mA). Close examination of the signal-to-noise ratio of individualband intensities allows the performance of quantitative footprintingdata analysis at time scales as low as 100 milliseconds (13). FIG. 5 bshows the densitometry scans through the 140 bp DNA fragment in variousexposure times to the X-ray beam. The top right insert representsvarious densitometry scans through a region of 10 bases in the middleregion of the gel obtained after different exposure times. The scansfrom bottom to top are of 0 (background), 100, 300, 500, 700 and 1000milliseconds. The integrated band intensities at 100 milliseconds show a3-fold increase in band intensity over the background and 10-foldincrease at 1 second.

Protein-DNA footprinting—The device of the invention was used to performprotein-DNA footprinting experiments on IHF protein bound to its targetDNA sequence. This complex has been characterized by both biochemicaland structural studies^((22, 30)). IHF binds tightly to three sitesalong a 34 bp region (see FIG. 6 a.). These sites produce threeprotection regions on the DNA target. The three binding sites of IHF onits target DNA region of 34 bp are marked in black boxes AI, AII andAIII. The interaction of IHF with its substrate DNA produces threeprotection regions. The 5′ end designated with asterisk marks theposition of ³²p labeling. FIG. 6 b shows IHF-DNA footprinting by bothFeEDTA and X-ray methods. 50 nM of IHF was incubated with its target DNAin 10 μl reaction volume at room temperature for 10 min. Sample wasexposed to FeEDTA cleavage (lane 1) or to X-ray beam for 1 second (lane4). Lane 2 is the control DNA and lane 3 is free DNA exposed to X-ray.The three binding sites resulted from the tight binding of IHF with the34 bp DNA region are marked AI, AII and AIII. All three sites areidentified in both lanes 1 and 2. The protein binding sites on thetarget DNA are marked as AI, AII, AIII, according to their correspondingsequences. These binding sites were analyzed by FeEDTA and in accordancewith the invention. The FeEDTA reaction was allowed to proceed for 2seconds; the X-ray experiments were conducted by irradiating the sampleswith single exposure of 500 milliseconds. FIG. 6 b lanes 1 and 4 showthat the three designated IHF binding sites can be clearly identified byFeEDTA and by the method of the invention. Furthermore, these resultsare in good agreement with recent studies by Dhavan et al., which reportthe footprinting pattern of the same IHF-DNA complex using thesynchrotron X-ray footprinting procedure⁽³¹⁾. FIG. 6 c showsdensitometry profiles of FIG. 6 b, lanes 3 & 4 (black and blue,respectively) showing a decrease in peak intensities upon binding of IHFon the DNA (FIG. 6 b, lane 4).

Comparisons of the normalized source spectrum and deposited spectrum onthe nucleic acid sample are presented for the synchrotron beam and forvarious X-ray anodes in FIG. 7. FIG. 7 shows the normalized photon fluxspectrum at the X-9 NSLS beam line compared with the normalized absorbedenergy distribution on a nucleic acid sample in aqueous solution. Thepeak energy on the sample falls between 11-12 KeV, in agreement with theresults shown in ref.⁽²⁷⁾. The error bars represent the simulationresults' standard deviation. The energy peak deposited from the X-raygenerator with Cu anode source (dotted line) falls very close to thepeak of the energy deposited from the X-9 NSLS. These results show thatthe Cu rotating anode energy is highly consistent with the peak energyabsorbed by the sample in the synchrotron source. However, in contrastto the wide range of energies applied to the sample by the synchrotronX-ray beam, the Cu anode spectrum provides a single or very narrowenergy range. Overall, these results show that the energy of theabsorbed photon, above a given value of photon flux, may be consideredas a limiting factor for effective DNA cleavage by X-rays in dilutesolutions. Therefore, the material of the target in the rotating anodegenerator should be carefully selected to obtain effective nucleic acidcleavage in short time scales. Nevertheless, up to 81% of the energyprovided by the Cu anode is absorbed in the DNA solution (Table 1),which improves the probability of DNA cleavage by the free —OH radicals.

1. A method for footprinting a nucleic acid molecule comprising: (a)placing the nucleic acid molecule in an environment in which —OHradicals are generated when the environment is irradiated with X-rays atan intensity of less than 10⁹ photons sec⁻¹ mm⁻² for an amount of timeless than 1,000 msec; (b) irradiating the environment with an X-ray beamproduced by a rotating Cu anode X-ray generator at an intensity of lessthan 10 ⁹ photons sec⁻¹ mm⁻² for an amount of time less than 1,000 msecso as to generate —OH radicals in the environment; (c) detectingfragmentation of the nucleic acid molecule.
 2. The method according toclaim 1 wherein the predetermined amount of time is from 100 to 300msec.